The study of molecular properties in complex chemical and biological samples requires sufficient spatial resolution as well as chemical selectivity. To achieve this, certain spectroscopic techniques have been combined with microscopy in order to investigate the properties of molecules in chemical and biological samples, including live cells. For example, chemically selective three-dimensional imaging of samples has been accomplished using two-photon fluorescence microscopy. In two-photon fluorescence microscopy, fluorophore labeled samples are imaged on a laser scanning microscope by a tightly focused femtosecond pulsed laser having a wavelength in the near-infrared region. However, photobleaching and chemical perturbations inherent in fluorophore labeling are limitations of the technique.
As another example, chemically selective three-dimensional imaging of samples has been accomplished used multiphoton vibrational microscopy based on coherent anti-Stokes Raman scattering (CARS). In CARS, a pump field, a Stokes field and a probe field interact with a sample to generate an anti-Stokes field at the frequency ωas=2ωp−ωs and wave vector kas=2kp−ks. In CARS microscopy, samples are imaged on a laser scanning microscope by a tightly focused pump laser pulse and Stokes laser pulse. (The pump field and probe field are generally derived from the same laser pulse.) Although CARS microscopy avoids the photobleaching problem and is a label-free method, the drawbacks of the technique include an intrinsically weak anti-Stokes signal and significant nonresonant background interference, both of which limit image contrast and spectral selectivity.